Not Applicable
The field of the present invention is protein biochemistry, in particular, the refolding of biologically active protein after dissolution of aggregated protein, which is often composed of denatured protein. In the present invention, refolding of such protein is carried out at relatively high pressures, relatively high protein concentration and desirably, in the presence of a relatively low concentration of at least one denaturant.
Existing methods for solubilizing and refolding protein aggregates and inclusion bodies into their native structure are discussed below. These methods include general processes and those developed for particular proteins. General processes are said to be useful for any protein aggregate, while xe2x80x9cspecificxe2x80x9d methods work for a single case, and effectiveness is not claimed other proteins.
All of the general processes described below utilize strong denaturing agents such as sodium dodecyl sulfate (SDS), urea, or guanidine hydrochloride followed by a dilution or dialysis step. These processes require large amounts of potentially hazardous chemicals and large volumes with dilution. With the combination of pressure, as taught by the instant invention, the amount of denaturing chemicals used are reduced by a factor of 10 or even eliminated. In addition, because the present process can operate at relatively high protein concentrations, the dilution step is no longer necessary.
U.S. Pat. No. 5,077,392 (1991) describes a process for activation of recombinant protein produced in prokaryotes, in which the aggregated proteins are dissolved in 4-8M guanidine hydrochloride or 6-10M urea. Once solubilized, the buffer is dialyzed to a pH between 1 and 4. Finally, the solution is diluted to provide a nondenaturing and oxidizing environment to allow for refolding.
U.S. Pat. No. 5,593,865 (1997) describes a process for activating recombinant disulfide bond-containing eukaryotic proteins after expression in prokaryote hosts. Inclusion body proteins are dissolved in a strong denaturing agent (6M guanidine hydrochloride) containing reducing agents. In the refolding step, proteins are introduced into an environment which is oxidizing and nondenaturing.
U.S. Pat. No. 4,677,196 (1987) also describes purification and production of biologically active proteins from insoluble inclusion bodies. This is a general method for recovering proteins from insoluble form includes dissolving the protein aggregates in SDS. Once dissolved, the protein solution is separated from SDS by column chromatography. In the absence of SDS, the protein can refold. Finally, the protein is eluted from the column. Urea has also been included in dissolved protein solutions. After anion exchange chromatography, the urea from the refolded protein solution is removed by dialysis.
U.S. Pat. No. 5,605,691 (1997) describes solubilization of inclusion body proteins using SDS and heat. Once in solution, proteins are refolded by first diluting the SDS and then dialyzing away the SDS to nondenaturing concentrations.
U.S. Pat. No. 4,659,568 (1997) describes a process for solubilization, purification and characterization of protein from insoluble protein aggregates or complexes and compositions of matter therefrom. The insoluble protein aggregates or inclusion bodies are layered on top of a urea step gradient (3M to 7M urea). As the samples are centrifuged, the aggregates move through the gradient until they dissolve. This method provides a means of determining the urea concentration at which the protein dissolves.
U.S. Pat. No. 5,728,804 (1995) describes a process in which denatured or aggregated proteins are suspended in a detergent-free aqueous medium containing 5-7 M guanidine hydrochloride and incubated overnight. Once suspended, the sample is contacted with sufficient cyclodextrin to assist in the refolding of the proteins. Finally, the cyclodextrin is removed by dialysis.
Turning now to patents for processes developed for particular proteins, U.S. Pat. No. 4,652,630 (1987) describes a method for producing active somatotropin. In this method, the aggregates or inclusion bodies are solubilized in a chaotrope (3M to 5M urea), and the pH is adjusted to allow complete solubilization. Then the conditions are modified to allow oxidation in the presence of a nondenaturing concentration of chaotrope.
U.S. Pat. No. 5,064,943 (1991) also describes a method for solubilizing and renaturing somatotropin, but it does not require the use of a chaotrope. Here, the pH is adjusted to between 11.5 and 12.5 and maintained for 5 to 12 hours. Under these conditions, somatotropin will solubilize and renature.
U.S. Pat. No. 5,023,323 (1991) describes a process for naturation of somatotropin (growth hormone) aggregates in which the aggregates are dissolved in a denaturing chaotrope (1M to 8M urea). The solubilization step is followed by exposing the sample to an oxidizing environment in the presence of a nondenaturing concentration of chaotrope.
U.S. Pat. No. 5,109,117 (1992) describes a method in which somatotropin aggregates are dissolved in the presence of an organic alcohol and chaotrope (1M to 8M urea). Then the solubilized proteins are renatured in a nondenaturing, oxidizing environment.
U.S. Pat. No. 5,714,371 (1998) provides a method for refolding aggregates of hepatitis C virus protease. Aggregates are solubilized in 5M guanidine hydrochloride. Second, a reducing agent is added to the solution, and the pH is adjusted to provide an acidic pH. Third, the denaturing agent is removed from the solution by dialysis, and finally the pH is raised to its starting point.
U.S. Pat. No. 4,923,967 (1990) describes a process specific for human interleukin-2. Protein aggregates are dissolved in 4-8M guanidine hydrochloride with a sulfitolyzing agent. Once the proteins are dissolved, the sulfitolyzing agent is removed by solvent exchange. Finally, the temperature is raised to precipitate out interleukin-2 in pure form. To allow refolding, precipitates are dissolved again in guanidine hydrochloride plus a reducing agent. Finally, the solution is diluted to refold proteins.
U.S. Pat. No. 5,162,507 (1992) describes a process for recovering purified, oxidized, renatured recombinant interleukin-2 from microorganisms. Insoluble interleukin-2 isolated from microorganisms is solubilized in 2M to 4M guanidine hydrochloride. The guanidine hydrochloride solution is then diluted until the proteins precipitate out of the solution. The precipitates are then redissolved in a guanidine hydrochloride solution. The proteins are then oxidized to reform native disulfide bonds. Finally, the solution is diluted and interleukin-2 remains in solution.
U.S. Pat. No. 4,985,544 (1991) describes a process for renaturing fish growth hormone. In this process, the aggregates or inclusion bodies are dissolved using guanidine, urea, SDS, acid or alkali. The reducing agent is then removed, and an oxidizing agent is added. Finally, the denaturing agent is removed to allow refolding.
U.S. Pat. No. 5,410,026 (1995) describes a method in which insoluble, misfolded insulin-like growth factor-1 (IGF-1) is refolded into an active conformation. Once IGF-1 is isolated, it is incubated with 1-3M urea or 1M guanidine hydrochloride until the aggregates are solubilized and refolded.
Other U.S. patents of interest include U.S. Pat. Nos. 5,708,148; 4,929,700 and 4,766,224.
Because management and disposal of biotechnological processing materials are time-consuming and costly, because protein aggregates and denatured proteins in pharmaceutical preparations are inefficacious and dangerous, and because poor yields of biologically active protein harms the economics of recombinant protein production, there is a longfelt need in the art for a process for the efficient preparation of properly fold, non-aggregated and fully active protein, especially that expressed by recombinant means.
The present invention provides methods for recovering a properly folded, biologically active protein from mixtures containing aggregates or inclusion bodies containing the protein, or from solutions containing the protein in a denatured state. Proteins in solution, even highly purified proteins, can form aggregates with themselves or with other proteins, upon storage, or during production processes that involve fluid transfers, contact with surfaces or by other non-specific causes. Such aggregation can lead to loss of biological activity and can be harmful in therapeutic uses. The methods of the present invention result in disaggregation of aggregates present in mixtures of aggregated and native protein, and in renaturation of denatured protein in solution.
The processes of the invention are applied to solutions or mixtures where total protein concentration is in the range of from about 0.001 mg/ml to about 500 mg/ml, preferably from about 0.1 mg/ml to about 25 mg/ml and most preferably from about 1 mg/ml to about 10 mg/ml. Optionally in the embodiment of the invention used to disaggregate a protein and depending upon the protein to be disaggregated, a chaotropic agent can be added to the protein mixture. However, the concentration of chaotropic agent is limited to that which permits retention of biological activity of the protein in its native form. As will be understood in the art, the concentration of chaotropic agent, if any, will be selected according to the particular protein to be disaggregated, based upon its sensitivity to the chaotropic agent. With these limitations in view, the chaotropic agent can be present at concentrations in the range from 0 M to about 8 M, most often in the range from 0 M to about 1.0 M. The concentrations employed will also differ depending on the particular chaotropic agent employed.
An important feature of the invention is the application of elevated pressure to the mixture to be disaggregated. Elevated pressure is applied in two stages, first to accomplish disaggregation, where the pressure to be applied ranges from about 0.25 kbar to about 12 kbar for a time sufficient for disaggregation to occur. The pressure to be applied is chosen with regard to the particular protein to be disaggregated. A second stage of incubation at elevated pressure is carried out to permit re-folding of the protein to its native, biologically active (at atmospheric pressure) conformation. Surprisingly, incubation of disaggregated or denatured protein at elevated pressure in the range from 0.25 kbar to about 3.3 kbar promotes reconstitution of the native, biologically active (at atmospheric pressure) protein, even though higher pressures promote denaturation. Preferably the incubation pressure for renaturation is in the range from about 2 kbar to about 3.3 kbar. The optimum pressure for renaturation depends on the individual protein to be treated, as will be understood in the art.
An oxidizing agent and a reducing agent can be optionally included in the renaturation solution, for example, dithiothreitol, in the concentration range from about 1 mM to about 50 mM, and reduced glutathione, in the concentration range from about 1 mM to 50 mM. The pressure of such redox reagents provides that disulfide bonds are both readily formed and reduced. The mixtures for disaggregating or renaturing protein can also comprise surfactants, non-specific protein stabilizing agents, buffering agents, ligands of the protein being refolded, and the like.
Another embodiment of the invention, also employs pressure-facilitated refolding of denatured protein. In this embodiment, denatured protein in solution is provided in the presence of denaturing amounts of a chaotropic agent. The protein concentration in solution is in the range from about 0.001 mg/ml to about 500 mg/ml, preferably from about 0.1 mg/ml to about 25 mg/ml, more preferably from about 1 mg/ml to about 10 mg/ml. The chaotropic agent ranges in concentration from about 2 M to about 8 M, depending upon the protein to be denatured and the chaotropic agent. The denatured protein solution is incubated at elevated pressure in the pressure range effective for facilitating renaturation, namely from about 0.25 kbar to about 3.3 kbar and preferably from about 2 kbar to about 3.3 kbar. While under pressure, the concentration of chaotropic agent is reduced by any suitable means, for example, by dilution or by dialysis, to a level sufficient to permit biological activity of the protein at atmospheric pressure. Incubation takes place for a time sufficient to permit re-folding of the protein which usually occurs within 24 hours, depending on the protein. At the end of the pressure incubation period, the pressure is reduced to atmospheric pressure. In both embodiments of the invention, redox agents, stability agents, surfactants and the like can be added to the solution as described, supra.
The processes of the invention can be carried out at any temperature between the freezing point of the aqueous medium (about 0xc2x0 C.) and the temperature at which biological activity is lost due to thermal denaturation. The upper limit will be somewhat different for each individual protein and will also be affected by the composition of the medium, pH, presence of stabilizing compounds and the like, as is known in the art. The preferred temperature for carrying out the process of the invention is within 20xc2x0 C. of the upper limit temperature. For the disaggregation of growth hormone, see Example 4, disaggregation and renaturation occurred more rapidly at 60xc2x0 C. (upper limit 80xc2x0 C.) than at room temperature.